This invention relates to techniques for analyzing body fluids and, more particularly, to a method and apparatus for determining crystallization properties of urine.
The mechanisms of renal stone formation are not fully understood. This is particularly true of calcium oxalate and calcium phosphate stones which are quite common. Some investigators have offered evidence in support of the theory that the dominating cause of calcium oxalate stone formation is supersaturation of urine with calcium ions and oxalate ions. These investigators have generally set forth ex-vivo evidence in support of this "supersaturation" theory, but the causes of chemical changes which lead to the stone formation are not fully explained. It would appear that if supersaturation of urine alone causes renal calculi, correction of this cause by limitation of ion excretion might be readily achievable. However, treatment to reduce supersaturation of crystallizable ions has not been found completely successful in arresting stone disease.
Other investigators have found evidence to support the theory that normal urine contains substances that inhibit the nucleation and/or growth of, or effect the solubility of, calcium oxalate. There have been certain indications, again not fully explained, that certain inhibiting substances found in normal urine are absent from the urine of stone formers.
It has been demonstrated that the particle size distribution of calcium oxalate crystals in fresh urine from recurrent stone formers is quite different than the distribution found in "control" non-stone-formers under the same conditions of dietary and fluid intake. For example, in an article entitled "Calcium Oxalate Crystalluria and Inhibitors of Crystallization in Recurrent Renal Stone-formers," which appeared in Volume 43 of "Clinical Science," it was reported that the crystals excreted by the controls were small and belong to a unimodal distribution, whereas those excreted by the stone-formers belong to a distribution which contained a second peak of much larger particles. In the same article, it was suggested that the urines of the controls contained an inhibitor of the growth and aggregation of calcium oxalate crystals in vitro and that the inhibitor was deficient in the urines of the recurrent stone-formers. While these findings are useful, it is necessary to enhance available data with information regarding the mechanisms of crystal growth and to measure the effects of the chemicals thought to be effective inhibitors, such as to determine the effectiveness of various treatments in producing the desired inhibiting action.
In an article entitled "The Inhibitory Effect of Urine on Calcium Oxalate Precipitation," which appeared in Volume 12, No. 6 of "Investigative Urology," there was described experiments in which a solution of natural urine in a buffered mixture containing calcium ions and oxalate ions had a measurable inhibitory effect on the formation of calcium oxalate precipitate. In these experiments the precipitate was recovered using a filter and calcium content was measured using flame photometry. In an attempt to determine the extent of inhibition of precipitation caused by the natural urine due to those urinary constituents known to effect calcium oxalate solubility, an "artificial urine," consisting of a mixture of nine salts and urea, was substituted for the natural urine and the experiments were repeated. In both cases (natural as well as artificial urine), it was found that the degree of inhibition of precipitate formation was related to the concentration of the urine in the solution. The "artificial urine" was found to have some inhibitory effect, but less than that of the same amount of natural urine.
The described prior art techniques, while useful, are limited by practical problems which arise in the attempt to isolate and determine the precise nature of the crystal forming and/or inhibiting effect of constituents in highly complicated natural urine. On the one hand, it would appear desirable to utilize relatively high concentrations of natural urine in performing experiments, since this apparently amplifies the effect to be measured and renders observations easier. On the other hand, due to the complex, variable, and still somewhat unknown nature of the constituents of natural urine and the reactions which occur in natural urine, the experimenter using relatively higher concentrations thereof must be concerned with "background reactions" which may tend to disturb measurements being taken by introducing unknown factors. A further practical limiting factor is that the amount of fresh natural urine from a particular subject being studied is limited by the subject's output, typically less than 2,000 milliliters per day under normal fluid intake conditions. Thus, techniques which require relatively large volumes of urine or numerous experiments to be performed using the urine of a given subject (each requiring a volume of urine) are rendered impractical.
In an article entitled "The Concept of a Continuous Crystallizer--Its Theory and Application to In Vivo and In Vitro Urinary Tract Models," by B. Finlayson, which appeared in Volume 9, No. 4 of "Investigative Urology," there is described an attempt at employing a "continuous crystallizer" for experiments wherein feed solutions including urine plus calcium and oxalate ions were crystallized under steady state conditions to determine properties of the crystallization process. The article cites a series of prior publications by Randolph et al which disclose various chemical engineering applications of particle balance to continuous crystallizers. In the Finlayson article, a "continuous crystallizer" is defined as any chamber continuously receiving a stream of supersaturated liquid and continuously ejecting a stream of liquid plus suspended particles. Finlayson reported employing a 550 milliliter continuous crystallizer into which were dripped the urine plus the salts including calcium and oxalate ions. After a number of system cycles had elapsed (i.e., cycles of mixing and removing to achieve a "steady state" of crystallization), measurements were taken on the reaction chamber contents by filtering to obtain the crystals and then making photomicrographs.
In the U.S. Pat. No. 4,025,307, there is disclosed a method and apparatus for determining crystallization properties, such as the particle kinetics, of urine, which overcame a number of the existing prior art problems. In the patent, feed solutions containing synthetic urine and an aliquot of natural urine are combined in a continuous crystallizer, and the particle densities or crystal populations of the contents of the crystallizer are measured. The particle kinetics, such as nucleation and growth rate of the crystals, can then be calculated as a function of the measured particle densities. In one form of the apparatus disclosed in the referenced patent, the volume of the continuous crystallizer is of the order of 100 milliliters, it having been found that this unusually small volume does not interfere with the obtainment of proper steady state continuous crystallization and the relatively accurate measurement of crystallization kinetics. Using the techniques disclosed in the referenced patent, the synthetic urine contributes necessary stability and control so that crystallization properties can be measured without undue "background" reactions, yet enough natural urine is present to yield sufficient quantities of the constituents being measured to provide adequate sensitivity. The two feed solutions, respectively contain calcium and oxalate ions in amounts sufficient to cause supersaturation in the continuous crystallizer and contain components of the type necessary to obtain weddelite-type crystals of calcium oxalate in the dihydrate form (this being the type of calcium oxalate crystals generally formed in human urine). Only relatively small amounts of natural urine are needed, and the relatively small size of the continuous crystallizer is advantageous in reducing the total amount of reagents (from the feeds) necessary to perform a given test.
While the advantages of the techniques disclosed in the U.S. Pat. No. 4,025,307 are apparent, applicant has found that various aspects thereof can stand improvement. For example, it is a property of a continuous crystallizer, such as a so-called "mixed suspension mixed product removal" crystallizer of the type preferred in the referenced patent, that reagents are continually fed to th crystallizer unit and overflow therefrom. This is done to obtain and maintain a "steady state" condition under which reactions can be studied. However, the total volume of reagents utilized in this procedure is typically many times the volume of the continuous crystallizer chamber. Accordingly, a substantial volume of reagents is required for each test, thereby compromising the practicality of the test procedure. Further, it would be advantageous to have more closely controllable and efficient means of maintaining the supersaturation level of calcium oxalate in the continuous crystallizer.
It is generally an object of the present invention to provide improvement over the technique set forth in the above-referenced U.S. Pat. No. 4,025,307.